Cardioprotective therapies based on enzymatic elimination of lipid peroxides by allene oxide synthase

ABSTRACT

The present invention relates to methods and compositions related to the use of enzymes of the allene oxide synthase family for reducing myocardial ischemia reperfusion injury associated with myocardial infarction, thrombolysis, angioplasty and coronary bypass surgery. The methods of the invention comprise administration of allene oxide synthases, which are capable of eliminating lipid hydroperoxides, as a potent cardioprotective agent. The invention is based on the discovery that administration of allene oxide synthase following severe global ischemia stimulates cardiac recovery.

SPECIFICATION

[0001] The present invention relates to methods and compositions relatedto the use of enzymes of the allene oxide synthase family for reducingmyocardial ischemia reperfusion injury associated with myocardialinfarction, thrombolysis, angioplasty and coronary bypass surgery. Themethods of the invention comprise administration of allene oxidesynthases, which are capable of eliminating lipid hydroperoxides, as apotent cardioprotective agent. The invention is based on the discoverythat administration of allene oxide synthase following severe globalischemia stimulates cardiac recovery.

BACKGROUND OF INVENTION

[0002] Ischemia, the clinical term for oxygen starvation, is the leadingcause of death in the world. The most common forms of ischemia leadingto death are caused by interruptions of blood flow. Heart disease,stroke, and severe blood loss from traumatic injuries are examples ofischemia that are responsible for over 50% of all deaths.

[0003] In clinical situations of ischemia, the immediate goal is torestore blood flow to the patient as quickly as possible. If blood flowis restored within a suitable time period, tissue damage can be averted.However, a significant delay in restoring blood flow leads to a secondcondition known as ischemia-reperfusion injury. Reperfusion injurybegins when harmful reactive oxygen species are formed following theischemic episode. Reactive oxygen species originate by complex,spontaneous free radical reactions that damage lipids, proteins and DNAand can eventually kill cells. Lipids in cellular membranes are theprimary early targets of attack by oxygen in the blood resulting information of lipid peroxides which are extremely cytotoxic (Kilgore K Sand Lucchesi B R. 1993, Clin Biochem 26:359-70; Kloner R A., 1993, J AmColl Cardiol 21:537-45; Huynh T T, et al., 1999, J Surg Res 84:223-32;Kesavulu M M, et al., 2001, Diabetes Res Clin Pract 53:33-9).

[0004] Ischemia-reperfusion injury can develop gradually after anischemic event and may cause irreversible damage to tissues. Clinicalexamples include cardiac contractile dysfunction, arrhythmias andirreversible myocyte damage (heart cell death) following myocardialinfarction (heart attack). Although no drugs are currently available fortreating ischemia-reperfusion injury, the damage caused byischemia-reperfusion injury is preventable and reversible if certaintreatments are administered in a timely fashion. The most clinicallyrelevant treatments involve administration of antioxidants. Endogenousantioxidants such as glutathione peroxidase (GPX), superoxide dismutase(SOD), and catalase act as a primary defense mechanism. Additionally,dietary vitamin E can play a secondary role in attenuatingischemia-reperfusion injury. Administration of exogenous antioxidantsupplements before or after reperfusion has shown some utility inexperimental systems (Abunasra H J et al., 2001, Eur J CardiothoracSurg. 20: 153-8; Cuzzocrea S, et al., 2001, Pharmacol Rev Mar 53:135-59;Dhalla N S, et al., 2000, Cardiovasc Res 47:446-56; Galang N, et al,2000, Toxicology 148:111-8; Hangaishi M, et al., 2001, Biochem BiophysRes Commun 285:1220-5; Kanamasa K, et al., 2001, Acta Cardiol 56: 181-6;Laurindo F R, et al., 1991, Circulation 83:1705-15; Li Q, 2001,Circulation 103: 1893-8; Prasad K, et al., 1996, Can J Cardiol12:1083-91; Rowland R T, et al.,1995, Surgery 118:446-52; Van Remmen H,et al., 2001, Am J Physiol Heart Circ Physiol 281 :H1422-32). However,SOD treatment has failed to show a beneficial effect in human clinicaltrials (Flaherty J T, 1994, Circulation 89: 1982-91).

[0005] Allene Oxide synthase serves as an antioxidant by rapidlyeliminating lipid hydroperoxides from the system (U.S. Pat. No.6,132,711; Pan Z, et al., 1998, J Biol Chem 273: 18139-18145). Thepresent invention provides a novel method for reducingischemia-reperfusion injury based on the administration of allene oxidesynthase.

SUMMARY OF THE INVENTION

[0006] The present invention relates to methods and compositions relatedto the use of enzymes of the family of allene oxide synthetases forreducing myocardial ischemia reperfusion injury associated withmyocardial infarction, thrombolysis, angioplasty and coronary bypasssurgery. The methods of the invention comprise administration of alleneoxide synthases as a potent cardioprotective agent against ischemiareperfusion injury. The invention is based on the discovery thatadministration of allene oxide synthase following severe global ischemiais capable of stimulating cardiac recovery.

DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention provides for compositions comprisingenzymes of the allene oxide synthase family and the use of suchcompositions for prevention and/or treatment of cardiovascular disease.Such cardiovascular diseases include, for example, myocardialinfarction, angina pectoris, thrombolysis, angioplasty, coronary bypasssurgery, vascular or myocardial ischemia, and related conditions aswould be known by those of skill in the art which involve dysfunctionof, or damage to, the heart or vasculature, and in particular, to tissuedamage related to accumulation of cytotoxic lipid hydroperoxides.

[0008] In addition, the compositions and methods of the presentinvention may be used to treat other types of ischemias relating toaccumulation of cytotoxic hydroperoxides in affected tissues. Suchischemias include but are not limited to mesenteric, brain (stroke),limb, optical and hepatic ischemias.

[0009] Many allene oxide synthases are known and, thus, can be producedby known methods from known starting materials, or may be prepared bymethods used to prepare allene oxide synthases as described in theliterature. For example, allene oxide synthase may be recombinantlyexpressed as described in U.S. Pat. No. 6,132,711. Alternatively, theallene oxide synthase may be commercially obtained. For purposes of thepresent invention, any member of the allene oxide synthase family may beutilized provided that the selected enzyme is capable of reducing thelevel of cytotoxic lipid hydroperoxides.

[0010] The present invention provides pharamaceutical compositionscomprising an allene oxide synthase in a pharmaceutically acceptablecarrier. Such carriers include any suitable physiological solution ordispersant or the like, such as saline or buffered saline. Carriers mayalso include, for example, any diluent, excipient, suspending agent,lubricating agent, adjuvant, vehicle, delivery system, emulsifier,absorbent, perservative, surfactant, colorant, flavorant or sweetner,antibacterial and antifungal agents, isotonic and adsorption delayingagents, and the like. Except insofar as any conventional media, carrieror agent is incompatible with the active ingredient, its use in thecompositions is contemplated.

[0011] The present invention is further directed to methods forprevention and/or treatment of cardiovascular disease. The inventionfinds particular utility in connection with surgical procedures such ascoronary bypass. In such instances, the allene oxide synthase wouldnormally be administered prior to, and if necessary, following theprocedure. The allene oxide synthase may also be used prior to or duringischemia reperfusion after a spontaneous ischemic event. Suchspontaneous events include but are not limited to subjects havingcoronary occlusion.

[0012] The method of the invention comprises the steps of administeringinto a subject prior to and/or at the time of ischemia reperfusioninjury an amount of allene oxide synthase capable of reducing the levelof lipid hydroperoxides and allowing the allene oxide synthase to comeinto effective contact with the tissue for reducing the ischemiareperfusion injury. The compositions of the invention may beadministered using a variety of different methods, including but notlimited to oral, pulmonary, parenteral (intramuscular, intraarticular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation or a fine mist), transdermal, nasal,vaginal, rectal, or sublingual routes of administration and can beformulated in dosage forms appropriate for each route of administration.

[0013] In preferred embodiments of the invention, the allene oxidesynthase is administered prior to ischemia reperfusion injury, typicallyby infusion of allene oxide synthase into the bloodstream.Alternatively, the allene oxide synthase is administered during ischemiareperfusion injury and prior to termination of tissue damage resultingfrom ischemia reperfusion injury. In instances, where the allene oxidesynthase is administered following commencement of ischemia reperfusioninjury, administration should be done within five days of suchcommencement, more preferably within twenty-four hours aftercommencement, and most preferably within one hour after commencement ofinjury.

[0014] Dosage of the compositions of the present invention to beadministered is determined with reference to various parameters,including the species of the subject, the age, weight, and diseasestatus and the particular physiological conditions requiring phenotypicalteration. The dosage is preferably chosen so that administrationcauses an effective result, as measured by molecular assays orphenotypic alteration. Such assays include, for example, measurement ofcardiac function, i.e., systolic and diastolic pressure, contractibilityand cardiac output.

6. EXAMPLE

[0015] Allene Oxide Syhthase Stimulates Recovery of Mechanical Functionof Hearts Following Ischemia

6.1. Material and Methods

[0016] Animals

[0017] Male Wistar rats (300-350 g) were obtained from Charles RiverLaboratories. Animals were housed two per cage and had free access tofood (standard rat chow) and water. Animals used in this study werecared for according the recommendations in The guide for the Care andUse of Laboratory Animals, National Institute of Health, Publ. No.85-23, revised 1986.

[0018] Ischemic Protocol

[0019] Hearts from previously CO₂-gassed and decapitated rats wereperfused and cannulated as Working hearts. Briefly, hearts were quicklyexcised, placed in ice-cold buffer, and immediately perfusedretrogradely via the aorta with Krebs-Henseleit buffer containing in(mM): 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, 0.5 EDTA, 25NaHCO₃, 11 glucose, 1.75 Ca²⁺ (pH 7.4, gassed with 95% O₂-5% CO₂).During this perfusion, the hearts were trimmed of excess tissue and theopenings of the left atria were cannulated. Hearts were then switched tothe working mode and perfused at a 11.5 mmHg left atrial fillingpressure and 80 mmHg aortic afterload in a recirculating buffer systemcontaining 11 mM glucose and 1.2 mM palmitate prebound to 3% bovineserum albumin. Hearts were perfused with this concentration of fattyacid since it mimics that observed in the clinical setting of ischemiaand reperfusion associated with cardiac surgery. After 15 minutes ofaerobic perfusion, hearts were subjected to 30 minutes of globalischemia by clamping off both left atrial filling and aortic afterloadline. Following ischemia, left atrial and aortic flow were restored andhearts reperfused for 30 minutes. Allene oxide synthase, when used, wasadded directly into the perfusate at the concentration of 250 μg 15minutes prior to the reperfusion period. At the end of reperfusion,hearts were removed from the cannulae and frozen in liquid N₂. Heartfunction was recorded using Transonic in-line flow probes (model T206)and a Dig Med Heart Performance Analyzer (model HPA-tau). Throughout theentire perfusion protocol, water jacketed chambers kept the temperatureof the perfusate at 37° C.

[0020] Statistical Analysis

[0021] For statistical comparison of group means, the Student t-lest wasused. A value of p<0.05 was considered significant. All data arereported as mean±SEM.

6.2. Results

[0022] Table 1 shows the effects of ADS on reperfusion recovery ofhearts following severe global ischemia. Under aerobic conditions,mechanical function was similar between hearts from control and alleneoxide synthase-treated groups. However, following 30 minutes of severeno-flow ischemia, allene oxide synthase was of benefit on recovery ofmechanical function. In fact, as early as 5 minutes into reperfusion,recovery of systolic pressure, end-diastolic pressure, contractility,and CO in allene oxide synthase-treated was significantly improvedcompared to non-treated hearts. Throughout the remaining of thereperfusion period, the benificial effects of allene oxide synthase onsystolic pressure, contractility, and cardiac output were observed.

[0023] A burst of oxygen free radicals typically observed uponreperfusion of ischemic hearts exposed to high levels of palmitate canresult in cardiac dysfunction. It is clear that allene oxide synthaseadded prior to the onset of reperfusion is of benefit. TABLE 1 Effectsof AOS on reperfusion recovery following ischemia. HR PSP EDP +dP/dt−dP/dt CO Group (beats/min) (mmHg) (mmHg) (mmHg/sec) (mmHg/sec) (ml/min)Aerobic perfusion 231 ± 11 104 ± 3 47 ± 2 1898 ± 160 1243 ± 100 41 ± 5control 223 ± 6 116 ± 4 44 ± 2 2089 ± 124 1372 ± 152 46 ± 2 +AOS 5 minreperfusion 174 ± 24 56 ± 18 33 ± 5 743 ± 429 455 ± 193 12 ± 9 control221 ± 11 112 ± 3^(†) 48: ± 1^(†) 2152 ± 157^(†) 1232 ± 159^(†) 40 ±2^(†) +AOS 10 min reperfusion 164 ± 3 75 ± 12 41 ± 2 795 ± 494 831 ± 10810 ± 5 control 210 ± 16 117 ± 7^(†) 42 ± 4 2149 ± 119^(†) 1340 ± 177^(†)42 ± 4^(‡) +AOS 20 min reperfusion 172 ± 19 82 ± 11 44 ± 3 1093 ± 359831 ± 132 17 ± 9 control 221 ± 7 119 ± 4^(†) 43 ± 1 2472 ± 101^(‡) 1482± 140^(†) 50 ± 4^(†) +AOS 30 min reperfusion 190 ± 18 85 ± 8 50 ± 3 1174± 241 727 ± 169 16 ± 9 control 234 ± 4^(†) 118 ± 4^(‡) 42 ± 1 2603 ±155^(‡) 1650 ± 124^(‡) 56 ± 3^(‡) +AOS

[0024] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims. Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

What is claimed:
 1. A method of treating ischemic injury comprisingadministering to a site of injury allene oxide synthase protein in anamount effective to prevent the ischemic injury.
 2. The method of claim1, wherein said ischemic injury is myocardial infarction.
 3. The methodof claim 1, wherein said ischemic injury is cerebrovascular stroke.
 4. Amethod of preventing ischemic injury comprising administering to thepotential site of injury allene oxide synthase protein in an amounteffective to prevent the ischemic injury.
 5. The method of claim 4,wherein said ischemic injury is myocardial infarction.
 6. The method ofclaim 4, wherein said ischemic injury is cerebrovascular stroke.
 7. Apharmaceutical composition comprising allene oxide synthase protein anda pharmaceutically acceptable carrier.